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Reconstitution of G Protein-Coupled Receptors into a Model Bilayer System: Reconstituted High-Density Lipoprotein Particles

  • Gisselle A. Vélez-Ruiz
  • Roger K. SunaharaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 756)

Abstract

Reconstituted high-density lipoprotein particles (rHDL) are powerful platforms used as a model phospholipid bilayer system to study membrane proteins. They consist of a discoidal-shaped planar bilayer of phospholipids that is surrounded by a dimer of apolipoprotein A-I (apoA-I). The amphipathic nature of apoA-1 shields the hydrophobic acyl chains of the lipids from solvent and keeps the particles soluble in aqueous environments. These monodispersed, nanoscale discoidal HDL particles are approximately 10–11 nm in diameter with a thickness that is dependent on the length of the phospholipid acyl chain. Reconstituted HDL particles can be assembled in vitro using purified apoA-1 and purified lipids. Investigators have utilized this model bilayer system to co-reconstitute membrane proteins, and take advantage of the small size and its monodispersion. Our laboratory and others have utilized the rHDL approach to study the behavior of G protein-coupled receptors. In this chapter, we describe strategies for the preparation of rHDL particles containing GPCRs in their monomeric form and discuss various methodologies used to analyze the reconstituted receptor function.

Key words

Apolipoprotein A-I High-density lipoprotein particles Receptor 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-Palmitoyl-2-oleoyl-sn-glycero-3-[phosphor-rac-(1-glycerol)] Monomer Oligomer 

Notes

Acknowledgments

This work is supported through funding of the National Institutes of Health (GM-068603 and GM-083118), the University of Michigan Biological Sciences Scholars Program and the Cellular and Molecular Biology Training Grant and the University of Michigan Rackham Merit Program.

References

  1. 1.
    Whorton, M. R., Bokoch, M. P., Rasmussen, S. G., Huang, B., Zare, R. N., Kobilka, B., and Sunahara, R. K. (2007) A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc Natl Acad Sci U S A 104, 7682–7. Copyright 2007 National Academy of Sciences USA.Google Scholar
  2. 2.
    Bayburt, T. H., and Sligar, S. G. (2003) Self-assembly of single integral membrane proteins into soluble nanoscale phospholipid bilayers. Protein Sci 12, 2476–81.PubMedCrossRefGoogle Scholar
  3. 3.
    Baas, B. J., Denisov, I. G., and Sligar, S. G. (2004) Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment. Arch Biochem Biophys 430, 218–28.PubMedCrossRefGoogle Scholar
  4. 4.
    Leitz, A. J., Bayburt, T. H., Barnakov, A. N., Springer, B. A., and Sligar, S. G. (2006) Functional reconstitution of Beta2-adrenergic receptors utilizing self-assembling Nanodisc technology. Biotechniques 40, 601–602, 604, 606.Google Scholar
  5. 5.
    Amin, D. N., and Hazelbauer, G. L. (2010) The chemoreceptor dimer is the unit of conformational coupling and transmembrane signaling. J Bacteriol 192, 1193–200.PubMedCrossRefGoogle Scholar
  6. 6.
    Raschle, T., Hiller, S., Yu, T. Y., Rice, A. J., Walz, T., and Wagner, G. (2009) Structural and functional characterization of the integral membrane protein VDAC-1 in lipid bilayer nanodiscs. J Am Chem Soc 131, 17777–9.PubMedCrossRefGoogle Scholar
  7. 7.
    Mi, L. Z., Grey, M. J., Nishida, N., Walz, T., Lu, C., and Springer, T. A. (2008) Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs. Biochemistry 47, 10314–23.PubMedCrossRefGoogle Scholar
  8. 8.
    Banerjee, S., Huber, T., and Sakmar, T. P. (2008) Rapid incorporation of functional rhodopsin into nanoscale apolipoprotein bound bilayer (NABB) particles. J Mol Biol 377, 1067–81.PubMedCrossRefGoogle Scholar
  9. 9.
    Tsukamoto, H., Sinha, A., Dewitt, M., and Farrens, D. L. (2010) Monomeric rhodopsin is the minimal functional unit required for arrestin binding. J Mol Biol 399, 501–11.Google Scholar
  10. 10.
    Whorton, M. R., Jastrzebska, B., Park, P. S., Fotiadis, D., Engel, A., Palczewski, K., and Sunahara, R. K. (2008) Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer. J Biol Chem 283, 4387–94.PubMedCrossRefGoogle Scholar
  11. 11.
    Kuszak, A. J., Pitchiaya, S., Anand, J. P., Mosberg, H. I., Walter, N. G., and Sunahara, R. K. (2009) Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2. J Biol Chem 284, 26732–41.PubMedCrossRefGoogle Scholar
  12. 12.
    Rogers, D. P., Roberts, L. M., Lebowitz, J., Datta, G., Anantharamaiah, G. M., Engler, J. A., and Brouillette, C. G. (1998) The lipid-free structure of apolipoprotein A-I: effects of amino-terminal deletions. Biochemistry 37, 11714–25.PubMedCrossRefGoogle Scholar
  13. 13.
    Rogers, D. P., Roberts, L. M., Lebowitz, J., Engler, J. A., and Brouillette, C. G. (1998) Structural analysis of apolipoprotein A-I: effects of amino- and carboxy-terminal deletions on the lipid-free structure. Biochemistry 37, 945–55.PubMedCrossRefGoogle Scholar
  14. 14.
    Segrest, J. P. (1977) Amphipathic helixes and plasma lipoproteins: thermodynamic and geometric considerations. Chem Phys Lipids 18, 7–22.PubMedCrossRefGoogle Scholar
  15. 15.
    Nolte, R. T., and Atkinson, D. (1992) Conformational analysis of apolipoprotein A-I and E-3 based on primary sequence and circular dichroism. Biophys J 63, 1221–39.PubMedCrossRefGoogle Scholar
  16. 16.
    Gorshkova, I. N., Liu, T., Kan, H. Y., Chroni, A., Zannis, V. I., and Atkinson, D. (2006) Structure and stability of apolipoprotein a-I in solution and in discoidal high-density lipoprotein probed by double charge ablation and deletion mutation. Biochemistry 45, 1242–54.PubMedCrossRefGoogle Scholar
  17. 17.
    Bhat, S., Sorci-Thomas, M. G., Alexander, E. T., Samuel, M. P., and Thomas, M. J. (2005) Intermolecular contact between globular N-terminal fold and C-terminal domain of ApoA-I stabilizes its lipid-bound conformation: studies employing chemical cross-linking and mass spectrometry. J Biol Chem 280, 33015–25.PubMedCrossRefGoogle Scholar
  18. 18.
    Thomas, M. J., Bhat, S., and Sorci-Thomas, M. G. (2006) The use of chemical cross-linking and mass spectrometry to elucidate the tertiary conformation of lipid-bound apolipoprotein A-I. Curr Opin Lipidol 17, 214–20.PubMedCrossRefGoogle Scholar
  19. 19.
    Li, H., Lyles, D. S., Thomas, M. J., Pan, W., and Sorci-Thomas, M. G. (2000) Structural determination of lipid-bound ApoA-I using fluorescence resonance energy transfer. J Biol Chem 275, 37048–54.PubMedCrossRefGoogle Scholar
  20. 20.
    Panagotopulos, S. E., Horace, E. M., Maiorano, J. N., and Davidson, W. S. (2001) Apolipoprotein A-I adopts a belt-like orientation in reconstituted high density lipoproteins. J Biol Chem 276, 42965–70.PubMedCrossRefGoogle Scholar
  21. 21.
    Koppaka, V., Silvestro, L., Engler, J. A., Brouillette, C. G., and Axelsen, P. H. (1999) The structure of human lipoprotein A-I. Evidence for the “belt” model. J Biol Chem 274, 14541–4.PubMedCrossRefGoogle Scholar
  22. 22.
    Gan, K. N., Smolen, A., Eckerson, H. W., and La Du, B. N. (1991) Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities. Drug Metab Dispos 19, 100–6.PubMedGoogle Scholar
  23. 23.
    Rogers, D. P., Brouillette, C. G., Engler, J. A., Tendian, S. W., Roberts, L., Mishra, V. K., Anantharamaiah, G. M., Lund-Katz, S., Phillips, M. C., and Ray, M. J. (1997) Truncation of the amino terminus of human apolipoprotein A-I substantially alters only the lipid-free conformation. Biochemistry 36, 288–300.PubMedCrossRefGoogle Scholar
  24. 24.
    Attie, A. D., Kastelein, J. P., and Hayden, M. R. (2001) Pivotal role of ABCA1 in reverse cholesterol transport influencing HDL levels and susceptibility to atherosclerosis. J Lipid Res 42, 1717–26.PubMedGoogle Scholar
  25. 25.
    Denisov, I. G., Grinkova, Y. V., Lazarides, A. A., and Sligar, S. G. (2004) Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size. J Am Chem Soc 126, 3477–87.PubMedCrossRefGoogle Scholar
  26. 26.
    Bayburt, T. H., Leitz, A. J., Xie, G., Oprian, D. D., and Sligar, S. G. (2007) Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins. J Biol Chem 282, 14875–81.PubMedCrossRefGoogle Scholar
  27. 27.
    Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001) Large-scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30, 544–546, 548, 550.Google Scholar
  28. 28.
    Lefkowitz, R. J., and Shenoy, S. K. (2005) Transduction of receptor signals by beta-arrestins. Science 308, 512–7.PubMedCrossRefGoogle Scholar
  29. 29.
    Kobilka, B. K. (1995) Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal Biochem 231, 269–71.PubMedCrossRefGoogle Scholar
  30. 30.
    Swaminath, G., Deupi, X., Lee, T. W., Zhu, W., Thian, F. S., Kobilka, T. S., and Kobilka, B. (2005) Probing the beta2 adrenoceptor binding site with catechol reveals differences in binding and activation by agonists and partial agonists. J Biol Chem 280, 22165–71.PubMedCrossRefGoogle Scholar
  31. 31.
    Alami, M., Dalal, K., Lelj-Garolla, B., Sligar, S. G., and Duong, F. (2007) Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA. EMBO J 26, 1995–2004.PubMedCrossRefGoogle Scholar
  32. 32.
    Boldog, T., Grimme, S., Li, M., Sligar, S. G., and Hazelbauer, G. L. (2006) Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties. Proc Natl Acad Sci U S A 103, 11509–14.PubMedCrossRefGoogle Scholar
  33. 33.
    Devanathan, S., Yao, Z., Salamon, Z., Kobilka, B., and Tollin, G. (2004) Plasmon-waveguide resonance studies of ligand binding to the human beta 2-adrenergic receptor. Biochemistry 43, 3280–8.PubMedCrossRefGoogle Scholar
  34. 34.
    Civjan, N. R., Bayburt, T. H., Schuler, M. A., and Sligar, S. G. (2003) Direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers. Biotechniques 35, 556–60; 562–3.Google Scholar
  35. 35.
    Forte, T., Norum, K. R., Glomset, J. A., and Nichols, A. V. (1971) Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: structure of low and high density lipoproteins as revealed by elctron microscopy. J Clin Invest 50, 1141–8.PubMedCrossRefGoogle Scholar
  36. 36.
    Lima, E. S., and Maranhao, R. C. (2004) Rapid, simple laser-light-scattering method for HDL particle sizing in whole plasma. Clin Chem 50, 1086–8.PubMedCrossRefGoogle Scholar
  37. 37.
    Segrest, J. P., Jones, M. K., Klon, A. E., Sheldahl, C. J., Hellinger, M., De Loof, H., and Harvey, S. C. (1999) A detailed molecular belt model for apolipoprotein A-I in discoidal high density lipoprotein. J Biol Chem 274, 31755–8.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of PharmacologyUniversity of Michigan Medical SchoolAnn ArborUSA

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